Carvedilol Induces Endogenous Hydrogen Sulfide Tissue Concentration Changes in Various Mouse Organs*
Bogdan WILIÑSKI, Jerzy WILIÑSKI, Eugeniusz SOMOGYI, Joanna PIOTROWSKA, Marta GÓRALSKA, and Barbara MACURA
Accepted May 19, 2011
WILIÑSKIB., WILIÑSKIJ., SOMOGYIE., PIOTROWSKAJ., GÓRALSKAM., MACURAB. 2011.
Carvedilol induces endogenous hydrogen sulfide tissue concentration changes in various mouse organs. Folia biologica (Kraków)59: 151-155.
Carvedilol, a third generation non-selective adrenoreceptor blocker, is widely used in cardiology. Its action has been proven to reach beyond adrenergic antagonism and involves multiple biological mechanisms. The interaction between carvedilol and endogenous
gasotransmitter hydrogen sulfide (H2S) is unknown. The aim of the study is to assess the influence of carvedilol on the H2S tissue level in mouse brain, liver, heart and kidney. Twenty eight SJL strain female mice were administered intraperitoneal injections of 2.5 mg/kg b.w./d (group D1, n = 7), 5 mg/kg b.w./d (group D2, n = 7) or 10 mg/kg b.w./d of carvedilol (group D3, n = 7). The control group (n = 7) received physiological saline in portions of the same volume (0.2 ml). Measurements of the free tissue H2S concentrations were performed according to the modified method of Siegel. A progressive decline in H2S tissue concentration along with an increase in carvedilol dose was observed in the brain (12.5%, 13.7% and 19.6%, respectively). Only the highest carvedilol dose induced a change in H2S tissue level in the heart an increase by 75.5%. In the liver medium and high doses of carvedilol increased the H2S level by 48.1% and 11.8%, respectively. In the kidney, group D2 showed a significant decrease of H2S tissue level (22.5%), while in the D3 group the H2S concentration increased by 12.9%. Our study has proven that carvedilol affects H2S tissue concentration in different mouse organs.
Key words: Hydrogen sulfide, carvedilol, adrenergic beta-antagonists, nitric oxide, mice.
Bogdan WILIÑSKI, Marta GÓRALSKA, Barbara MACURA, Department of Human Developmen- tal Biology, Jagiellonian University Medical College, Kopernika 7, 31-034 Kraków, Poland.
E-mail: bowil@interia.pl
Jerzy WILIÑSKI, I Department of Cardiology and Hypertension, Jagiellonian University Medical College, Kopernika 17, 31-501 Kraków, Poland.
E-mail: putamen@interia.pl
Eugeniusz SOMOGYI, Joanna PIOTROWSKA, Department of Inorganic and Analytical Chemis- try, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland.
E-mail: jpiotrowl@cm-uj.krakow.pl
Carvedilol, a third generation non-selective ad- renoreceptor blocker, is widely used in cardiology and general practice in the treatment of chronic diseases like congestive heart failure and arterial hypertension (CHAKRABORTY et al. 2010). The action of carvedilol has been proven to reach be- yond adrenergic antagonism and comprises i.a. an- tioxidant activity, calcium channel blockade and nitric oxide (NO) production enhancement (KOSTKA- -JEZIERNY& TYKARSKI2009). On the other hand, endogenously formed ‘gasotransmitter’ hydrogen sulfide (H2S) has been identified as a crucial regu- lator of circulatory, nervous, gastrointestinal and excretory systems. Altered production of H2S was observed in arterial hypertension, myocardial ische-
mia and atherosclerosis (£OWICKA& BE£TOWSKI
2007). The interaction between carvedilol and en- dogenous H2S is unknown.
The aim of the study is to assess the influence of carvedilol on the endogenous tissue H2S concen- trations in mouse brain, heart, liver and kidney.
Material and Methods
Animals
Twenty eight SJL strain female mice (11-12 week old individuals) of approximately 20 g weight were involved in the study. The animals were housed under standard laboratory conditions and had free
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*Supported by the grant No. K/ZBW/000175 from the Jagiellonian University Medical College.
access to water and food. They were kept at a tem- perature of 22-24°C with a light/dark cycle of 12 h.
Study protocol
A non-selective lipophilic â-blocker / á-1 blocker carvedilol (Avedol, Polpharma, Poland) was dis- solved in physiological saline. The study design comprised intraperitoneal injections of 2.5 mg per kg of body weight of carvedilol daily (group D1, n = 7), 5 mg per kg of body weight of carvedilol daily (group D2, n = 7) or 10 mg per kg of body weight of carvedilol daily (group D3, n = 7) for 5 consecutive days at the same time of day (10:30 am), each ad- ministration consisted of 0.2 ml of the solution.
The control population (n = 7) received intraperi- toneally physiological saline in portions of the same volume. The individuals were randomly as- signed to each group. The animals tolerated the ap- plied doses of carvedilol well and remained in good condition till the end of the experiment.
Measurements of the free tissue H2S concentration were performed by the use of the modified method of Siegel (SIEGEL 1965; SOMOGYI et al. 2008).
The study has been performed in accordance with the guidelines for the care and use of laboratory animals accepted by Bioethical Committee of the Jagiellonian University Medical College (Kraków, Poland).
Tissue sample preparation
Two hours after the last drug or physiological sa- line injection the animals were killed by cervical dislocation, their brains, hearts, livers and kidneys were quickly removed, homogenized with 0.01 mol/l sodium hydroxide (NaOH): brain tissue in propor- tion of 1 to 4, liver and kidney of 1 to 5 and heart of 1 to 10 and frozen. Then 50% trichloroacetic acid (TCA) was added (0.5 ml to 2 g of brain or liver samples in tight capsules of 3 ml and 0.25 ml to 1 g of heart or kidney sample in tight capsules of 2 ml), the suspension was shaken and centrifuged. Sub- sequently, 1.5 ml brain or liver and 0.75 ml heart or kidney supernatant samples were moved to 2 ml tight capsules with 0.15 ml or 0.075 ml of 0.02 mol/l N,N-dimethyl-p-phenyl-diamine sulfate in 7.2 mol/l hydrochloric acid (HCl), then 0.15 ml or 0.075 ml of 0.03 mol/l iron (III) chloride (FeCl3) in 1.2 mol/l HCl portions were added, respectively. After 20 minutes in darkness the content was shaken for 1 minute with 1 ml of chloroform.
H2S tissue concentration measurements Absorbance was measured at 650 nm with the Varian Cary 100 spectrophotometer. A standard curve was prepared with an iodometrically determined
0.0001 mol/l sodium sulfide (Na2S) solution. For each group of animals four concurrent analyses of each analyzed tissue type were performed.
Statistical analysis
Statistical analysis was performed within the R Environment by the Student’s t-test and univariate analysis of variance (ANOVA). Statistical signifi- cance was considered when P<0.05.
Results and Discussion
Progressive H2S tissue concentration decline was observed in the brain along with a rising carvedilol dose (by 12.5%, 13.7% and 19.6%, respectively).
In the heart only the highest carvedilol dose in- duced H2S tissue level change, but the increase was spectacular, reaching 75.5%. In the liver, me- dium and high doses altered the H2S concentration – 5 mg/kg b.w./d of carvedilol increased the H2S level by 48.1% and 10 mg/kg b.w/d by 11.8%. In the kidney, the group D2 showed a significant de- crease of H2S tissue level (22.5%), while in the D3 group H2S concentration increased by 12.9%. In the variance analysis for each tissue type only H2S concentration changes within the heart and brain were statistically significant (Table 1). Noteworthy, each organ has different metabolism, paracrine and endocrine regulation, and specific transmitter interac- tions, thus variable changes of H2S concentrations only confirm this complexity and heterogeneity.
Carvedilol’s therapeutic actions could not be fully explained by adrenoreceptor blockade. Nu- merous studies have provided evidence that carve- dilol has various other properties including antioxidant action, calcium channel antagonism, anti-inflammatory actions: fall in interleukin-1 (IL-1), interleukin-6 (IL-6), c-reactive protein (CRP) and tumor necrosis factor-á (TNF-á), direct inhi- bition of transcription factors like NF-êB, low den- sity lipoproteins (LDL) oxidation; stabilization of atherosclerotic plaques by decreasing intercellular adhesion molecule-1 (ICAM-1) and activity of ma- trix metalloproteinases 2 and 9 (MMP-2, MMP-9);
prevention of endothelial and myocardium apop- tosis, reversal of cardiac remodeling in chronic heart failure and endothelin-1 (ET-1) suppression (BELLENGERet al.2004; KALINOWSKIet al.2003;
KOSTKA-JEZIERNY& TYKARSKI2009; ROMEOet al.
2000; RUFFOLOet al.1993). Moreover, the major proportion of carvedilol’s biological action, espe- cially regarding hypotensive and vascular effects, seems to be mediated by NO, whose level rises due to endothelial nitric oxide synthase (NOS) stimu- lation (AFONSOet al.2006).
H2S is endogenously formed from L-cysteine in several enzymatic reactions catalyzed by cystathio- nine â-synthase (CBS), cystathionine ã-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST), and in non-enzymatic pathways in many tissues.
Cytoplasmatic bound sulfur is postulated to absorb and store exogenously applied and endogenously produced H2S which is released from the bound sulfur pool in the presence of physiologic concen- trations of glutathione and cysteine in slightly al- kaline conditions (ISHIGAMIet al. 2009). H2S acts as a ‘gasotransmitter’ and serves as a co-modulator of various physiological and pathophysiological processes such as regulation of vascular tone, myocardial contractility, neurotransmission and perception (FIORUCCIet al.2006; SHIBUYAet al.
2009). Its biological action comprises numerous intracellular mechanisms including adenosine tri- phosphate (ATP)-sensitive potassium channels (KATP) stimulation, sulfhydration of different pro- teins and maintaining protein–SH groups in the re- duced state, reaction with reactive oxygen and nitrogen species (ROS and RNS) (£OWICKA &
BE£TOWSKI2007; SUNet al.2008). H2S interacts with carbon monoxide (CO) and nitric oxide (NO) in a number of ways including affecting each other’s synthesis and biological responses within target tissues. All these three gases bind to haemo- globin and impede mitochondrial oxidative phos- phorylation by inhibiting cytochrome c oxidase (LIet al. 2009). Analogically to carvedilol, H2S decreases IL-6, TNF-á levels, reduces the activa- tion of NF-êB complex and the activity of MMP-2 and MMP-9 (OH et al. 2006; SEN et al. 2009;
SODHAet al.2009).
Our study has shown that carvedilol’s action in- volves H2S, probably via its production rate altera- tion and release with possible NO share as one of the mechanisms. Some of the biological effects of the beta-blocker and H2S are common. It is un- known whether H2S mediates any of them and to what extent carvedilol’s biology is dependent on the messenger, since research dedicated to the is- sue has not been done. In the heart, the cardiopro- tective effects of H2S have been demonstrated to involve opening of KATPchannels as well as effects of preserving mitochondrial structure and function (ELRODet al.2007). It might be an accessory ef- fect to NO beneficial impact on intracellular tran- scription factors resulting in increased expression of cardioprotective proteins like superoxide dismu- tase (SOD), inducible NOS (iNOS), cyclooxygenase-2 (COX-2) and heat shock proteins (DAWN& BOLLI
2002). In hepatology, carvedilol appears to be a potentially viable option for treating portal hyper- tension along with angiotensin-converting en- zyme inhibitors (ACEI) (HEMSTREET2004). H2S regulates perfusion pressure in normal and cir- rhotic liver in a NO-independent manner; NO and H2S are released by different cellular sources and their hemodynamic effects involve different cellu- lar targets. H2S generation in cirrhosis is decreased due to a reduced expression/activity of CSE in he- patic stellate cells (HSC) – one of the main sources of H2S in the liver (FIORUCCIet al.2005). In our previous studies a tissue specific ACEI ramipril also enhanced the H2S tissue level in the liver, heart and kidney (WILIÑSKIet al.2010; WILIÑSKI
et al.2008). In kidneys H2S has been recognized as an important regulator of renal function affecting both vascular and tubular actions (XIAet al.2009).
Some studies with carvedilol demonstrate attenu- Table 1 Hydrogen sulfide (H2S) tissue concentration in mouse brain, heart, liver and kidney follow- ing the administration of 2.5 mg/kg b.w. per day, 5 mg/kg b.w. per day or 10 mg/kg b.w. per day of carvedilol (groups D1, D2 and D3 respectively)
H2S tissue concentration
(Fg/g) Control group
(n = 7) D1
(n = 7) D2
(n = 7) D3
(n = 7) ANOVA
P
Brain 2.55 ± 0.04 2.23 ± 0.05** 2.20 ± 0.05** 2.05 ± 0.05*** <0.001
Heart 6.78 ± 0.06 5.91 ± 0.15 6.61 ± 0.14 11.90 ± 0.12*** <0.001
Liver 4.14 ± 0.04 4.17 ± 0.08 6.13 ± 0.14*** 4.63 ± 0.10** 0.22
Kidney 8.66 ± 0.19 8.45 ± 0.17 6.71 ± 0.08*** 9.78 ± 0.18*** 0.21
*P<0.05 for given group vs control group, **P<0.01 for given group vs control group, ***P<0.001 for given group vs control group
ated increases in albuminuria as well as reduction in cardiovascular events in chronic kidney disease patients with hypertension (BAKRIS et al.2006).
Carvedilol also protected against the renal mito- chondrial toxicity induced by cisplatin and daunorubicin-induced cardiotoxicity and nephro- toxicity in rats (AROZALet al.2010; RODRIGUES
et al.2010).
H2S acts as a neuromodulator as well as an intra- cellular messenger in the central nervous system.
Its perturbed metabolism has been investigated in many neurological disorders including Alzheimer’s disease (QUet al.2008). Recently, carvedilol was found to improve neuronal transmission and at- tenuate brain oligomeric beta-amyloid content and cognitive deterioration in two mouse models of Alzheimer’s disease (WANGet al.2010). Analogi- cally, non-steroidal anti-inflammatory drugs were also demonstrated to exert some anti-amyloidogenic effects and to affect H2S biology (BILSKAet al.
2010; SREBROet al.2006).
In conclusion, carvedilol affects the H2S tissue concentrations in mouse brain, heart, liver and kid- ney. The involvement of H2S makes the biological action of carvedilol more complex and opens new fields for investigation for both of them in neurol- ogy, cardiology, hepatology and nephrology. Several biotechnology companies are already developing H2S-based therapeutic compounds, and there are ongoing clinical trials investigating the therapeu- tic potential of H2S (PREDMORE& LEFER2010).
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